The specimens were embedded in paraffin and cut into 5-μm sections. The sections were deparaffinized and incubated for 10 min with PBS containing 3% H2O2 to block endogenous peroxidase activity. Heat-induced antigen heat retrieval was carried out for 10 min in a citrate buffer (pH 6.0) at 100°C. Sections were blocked for 1 h with 5% normal goat serum in PBS at room temperature and then incubated with anti-PCNA, anti-Bax, anti-Bcl-2, and anti-Ki67 antibodies overnight at 4°C. The next day, sections were incubated with their respective secondary antibodies for 1 h at room temperature. The peroxidase label was determined with diaminobenzidine hydrochloride (DAB, Sigma). Negative controls were incubated with pre-immune serum instead of the primary antibodies. Slides were counter-stained with hematoxylin and mounted with Permount.
Diaminobenzidine hydrochloride dab
Manufactured by Merck Group
Sourced in United States
Diaminobenzidine hydrochloride (DAB) is a chromogenic substrate used in immunohistochemistry and immunocytochemistry for the detection of target proteins. It produces a brown colored precipitate at the site of the antibody-antigen reaction, allowing for the visualization of the target protein.
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2 protocols using diaminobenzidine hydrochloride dab
1
Immunohistochemical Analysis of Cell Proliferation
2
Detailed Cultivation and Identification of H. pylori Strains
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Sydney strain 1, H. pylori SS1 (VacA+, CagA-), and Hp49503 (VacA+, CagA+) strains were purchased from the Department of Pathogenic Biology, School of Medicine, Shanghai Jiao Tong University. Brucella Broth was provided by the Municipal Center for Disease Control and Prevention of Shanghai. Agar powder was purchased from Oxoid (UK). Simple culture tank with ventilation was prepared by the Shanghai Academy of Life Sciences, Chinese Academy of Sciences. Others included Gram staining kit (Shanghai Kang'en Biotech Co. Ltd), H. pylori detection kit (urease method) (Zhuhai Lituo Biotech Co. Ltd), diaminobenzidine hydrochloride (DAB) (Sigma, USA), mixed gas (85% N2, 10% CO2, 5% O2) (Shanghai Youjiali Liquid Nitrogen Co. Ltd.), high-pressure steam sterilizer (SANYO, Japan), ultraclean workbench (Suzhou Yongjiu Pressure Regulator Factory), CX31 microscope (Olympus, Japan), electronic balance (Sartorius, Germany), RM2145 slicer (Leica, Germany), ultralow temperature refrigerator (SANYO, Japan), CD177 antibody (ab8092; Abcam), UV inhibitor (Roche Diagnostics), UV UNIV MULT HRP (Roche Diagnostics), carbolic acid red dye solution (Beijing Leigen Biotech Co. Ltd), ultrapure water meter (Millipore, USA), electric thermostatic water bath (Shanghai Yiheng Co. Ltd), and benchtop horizontal centrifuge (Eppendorf, Germany).
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